Journal of microbiological methods最新文献

{"title":"Beyond cold chain: A cost-effective ambient temperature storage and transportation of viable bacteria","authors":"Pratibha Mambatta Shankaranarayanan,&nbsp;Saranya Kanukollu,&nbsp;Bhavya Bharti,&nbsp;Avinash Shankar,&nbsp;Sneha Palpandi,&nbsp;Anand Babu Vangala,&nbsp;Elango E. Murugaian,&nbsp;Rajanikanth Vangala","doi":"10.1016/j.mimet.2025.107147","DOIUrl":"10.1016/j.mimet.2025.107147","url":null,"abstract":"<div><div>Freeze-drying and cryopreservation are the most common methods for microbial preservation. instaPRESERVE MicrobeSafe (IP-MIS), an innovative alternative for maintaining high bacterial viability and recovery at room temperature for up to 56 days. This study validates IP-MIS as an effective and reliable solution for short-term storage without the need for cold chain.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107147"},"PeriodicalIF":1.7,"publicationDate":"2025-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143970254","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Design and validation of universal and taxon-specific 16S rRNA qPCR primers for detection of freshwater harmful algal bloom-forming cyanobacteria","authors":"Ping Gong ,&nbsp;Anna K. Antrim ,&nbsp;Alyxandra S. Cicerrella ,&nbsp;Seung Ho Chung ,&nbsp;Xiao Luo ,&nbsp;Natalie D. Barker ,&nbsp;Emily Cooley ,&nbsp;Qing Ji","doi":"10.1016/j.mimet.2025.107146","DOIUrl":"10.1016/j.mimet.2025.107146","url":null,"abstract":"<div><div>Freshwater harmful algal bloom-forming cyanobacteria have become an increasingly prominent global concern from both environmental and human health perspectives. To enable and facilitate timely decision making in taking preventative and mitigative measures, rapid, accurate, sensitive and quantitative tools are needed for the detection and monitoring of toxin-producing cyanobacteria. Here we report the development of taxon-specific quantitative polymerase chain reaction (qPCR) primers capable of distinguishing 10 cyanobacterial genera or clade from non-target groups as well as a new set of universal primers capable of amplifying all cyanobacteria species. When evaluated by 4 stringent metrics and primer-template mismatches, these de novo designed qPCR primers outperformed published primers in amplifying the 16S rRNA gene of their target strains among the collection of 16 in-house cyanobacterial strains belonging to 10 genera. The 10 best-performing designed primers were validated using field samples from three field locations with historically documented HAB events. The field validation results corroborated with both microscopic observations and Nanopore sequencing of full-length 16S amplicons. Our study demonstrated the effectiveness of our design-screen-evaluation-validation pipeline in developing taxon-specific qPCR primers for detecting and quantifying group-specific target populations and their promising application to field HABs samples. With the advancement of massive parallel sequencing technologies and bioinformatic tools, a community-wide 16S full-length sequencing run can provide a panoramic view of the genetic diversity and site-specific variant info about the target taxa of interest, enabling the fast development of new taxon-specific qPCR assays and their refinement or modification to adapt to site-specific genetic variations in field samples.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107146"},"PeriodicalIF":1.7,"publicationDate":"2025-05-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144029243","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"The choice of DNA extraction protocol affects the quantification of gut microbiomes in two passerines","authors":"Hélène Dion-Phénix ,&nbsp;Geneviève Bourret ,&nbsp;Anne Charmantier ,&nbsp;Steven W. Kembel ,&nbsp;Denis Réale","doi":"10.1016/j.mimet.2025.107144","DOIUrl":"10.1016/j.mimet.2025.107144","url":null,"abstract":"<div><div>There is ever increasing need for the robust characterization of the microbial communities of wild animals. DNA extraction from bird feces is challenging and to date, no protocol has proven to be efficient with all bird feces samples. Thus, there is a need to test different extraction protocols for a variety of bird species. We compared five commercial kits and four protocols to extract DNA from black-capped chickadee and blue tit feces. We found that all kits and methods allowed the study of the bacterial microbiota of black-capped chickadee feces, but the choice of kit influenced the measured diversity and composition of microbiota communities. Only two kits out of five allowed the recovery of DNA from blue tit feces. We recommend using PowerSoil by Qiagen or QuickDNA by Zymo Research with black-capped chickadee feces, and MagMAX by Fisher for blue tit feces. Our study highlights the difficulty of extracting microbial DNA from bird feces, points out the limits of comparing bacterial communities across studies using different methods, and proposes optimized efficient protocols to extract microbial DNA from feces of two commonly studied bird species for the study of bacterial microbiota.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107144"},"PeriodicalIF":1.7,"publicationDate":"2025-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143946951","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Investigation of cagA, dupA and babA genes among clinical Helicobacter pylori isolates collected from patients suffering from H. pylori gastric disease using real-time PCR","authors":"Zahra Naderi ,&nbsp;Reza Mirnejad ,&nbsp;Mansour Bayat","doi":"10.1016/j.mimet.2025.107143","DOIUrl":"10.1016/j.mimet.2025.107143","url":null,"abstract":"<div><h3>Background</h3><div><em>Helicobacter pylori</em> (<em>H. pylori</em>) infection causes chronic gastritis and can lead to severe gastrointestinal diseases, including stomach ulcers, stomach cancer, and mucosa-associated lymphoid tissue (MALT) lymphoma. This infection affects about half of the world's population, with varying prevalence based on location and hygiene standards. Determining the importance of <em>H. pylori</em> pathogenic genes in predicting clinical outcomes is crucial, especially considering high rates of stomach cancer in Middle Eastern and Asian populations. The main aim of this study was to determine the frequency of the <em>babA</em>, <em>cagA,</em> and <em>dupA</em> pathogenic genes in <em>H. pylori</em> isolates obtained from patients with gastrointestinal diseases.</div></div><div><h3>Methods</h3><div>A urease test was conducted on 111 biopsies from the antrum of patients undergoing endoscopies at Tehran hospitals. Following this, an extraction kit was used, and the Real-time PCR technique determined the frequency of the <em>babA</em>, <em>cagA</em>, and <em>dupA</em> genes.</div></div><div><h3>Results</h3><div>Out of 111 stomach biopsies, 70 tested positive for <em>H. pylori</em>. Molecular analysis showed that the frequency of <em>babA</em>, <em>cagA</em>, and <em>dupA</em> genes was 51 (72.8 %), 35 (50 %), and 26 (37.14 %), respectively.</div></div><div><h3>Conclusion</h3><div>The results showed that <em>babA</em> and <em>cagA</em> genes were identified with a higher abundance among patients and the findings suggest that the presence of these genes could be considered a risk indicator for exacerbation of gastrointestinal disease. Additionally, the <em>dupA</em> gene was found with lower frequency, which is less likely to play a significant role in pathogenicity compared to other genes. This study could help develop more effective prevention and treatment methods in the future.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107143"},"PeriodicalIF":1.7,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143921781","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Novel Disk diffusion Assay for Filamentous Fungi Susceptibility to antifungals (DAFFS)","authors":"Salustra S. Urbin,&nbsp;Hailey Casey,&nbsp;Anna P.C. Price,&nbsp;Annette E. LaBauve,&nbsp;Carolyn L. Fisher","doi":"10.1016/j.mimet.2025.107145","DOIUrl":"10.1016/j.mimet.2025.107145","url":null,"abstract":"<div><div>Filamentous fungi are recognized as significant human pathogens, particularly in immunocompromised populations. The rise in antifungal resistance and geographical expansion due to environmental changes, including climate change, further complicates treatment efforts. Existing antifungal susceptibility testing methods often face challenges when applied to filamentous fungi. Variability in results, extended incubation times, and difficulties in standardization can compromise broth dilution and disk diffusion assays. Here, we present an improved method, the <strong>D</strong>isk diffusion <strong>A</strong>ssay for <strong>F</strong>ilamentous <strong>F</strong>ungi <strong>S</strong>usceptibility to antifungals (DAFFS), designed to overcome current challenges in antifungal susceptibility testing for filamentous fungi. DAFFS integrates optimized elements from standard methods for more efficient screening of filamentous fungi for susceptibility to established and novel antifungals. This approach offers a streamlined, reliable alternative for assessing fungal drug susceptibility by addressing the limitations of current testing methods.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107145"},"PeriodicalIF":1.7,"publicationDate":"2025-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143921783","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"β-cyclodextrine coated zinc oxide decorated graphene oxide nano hybrid for photocataylytic, antibacterial, and antifungal applications","authors":"Arti Hadap ,&nbsp;Aseel A. Kadhem ,&nbsp;Neeta Gupta ,&nbsp;Rey Y. Capangpangan ,&nbsp;Arnold C. Alguno ,&nbsp;Pratik Kumar Jagtap ,&nbsp;Akhil Saxena","doi":"10.1016/j.mimet.2025.107139","DOIUrl":"10.1016/j.mimet.2025.107139","url":null,"abstract":"<div><div>Polymer encapsulated zero dimensional/2D hybrids are considered as an efficient multipurpose nanohybrids. Herein, we report a two-step fabrication of β-cyclodextrine (β-CD) stabilized zinc oxide nanoparticles supported over 2D graphene oxide (GO) sheets. The nanocomposite showed band gap in the semiconductor region (∼5.6 eV) which has been confirm by Tauc plot derived from UV–visible spectrum. Fourier transformed infrared (FTIR) spectra of the composites implied its polymer attachment and partial reduction of GO. Time dependent dye degradation has been performed in present of light which showed significant efficacy within 3 h. Moreover, for bacterial and antifungal versatility, the nanohybrids has been tested against both the gram positive and gram negative bacterial strain resulting death of the strains due to generation of reactive oxygen species from the nanohybrid. To effectively prevent bacterial and mold growth, propagation, and survival in medical devices, these results suggest that the nanohybrid could be an ideal alternative for antibacterial, antifungal, and catalytic surface coatings which have an obvious impact not only in biomedical sectors but also in durable fabric coating applications.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107139"},"PeriodicalIF":1.7,"publicationDate":"2025-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144005105","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Development of polymerase chain reaction assays specific for individual pathogenic Aeromonas species","authors":"Yoshihiro Uno ,&nbsp;Kazumi Umeki ,&nbsp;Akiteru Yamada ,&nbsp;Yuki Hashikura ,&nbsp;Hajime Nomura ,&nbsp;Masahiro Hayashi ,&nbsp;Kunihiko Umekita","doi":"10.1016/j.mimet.2025.107142","DOIUrl":"10.1016/j.mimet.2025.107142","url":null,"abstract":"<div><div>The genus <em>Aeromonas</em>, a group of Gram-negative bacilli, inhabits a wide variety of aquatic environments, including rivers, lakes, and coastal seas. Among <em>Aeromonas</em> species, <em>A. hydrophila</em>, <em>A. caviae</em>, <em>A. veronii</em>, and <em>A. dhakensis</em> are clinically important species that cause infections in humans, leading to diarrhea associated with gastroenteritis, necrotizing fasciitis, and sepsis. <em>A. dhakensis</em> is a newly established species that was reclassified in 2013, and it is considered more pathogenic than other <em>Aeromonas</em> species. However, accurate identification of <em>Aeromonas</em> species by biochemical characteristics, mass spectrometry, and 16S rRNA gene analysis can sometimes be difficult. In this study, 24 strains were classified into nine species by homology and phylogenetic analysis of the RNA polymerase B subunit gene (<em>rpoB</em>), the gold standard method for identifying <em>Aeromonas</em> species. The results of 16S rRNA gene analysis were consistent with those of <em>rpoB</em> analysis for all strains. The results of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry were consistent with those of <em>rpoB</em> analysis for all strains excluding <em>A. dhakensis</em>; however, all five <em>A. dhakensis</em> strains were misidentified as other species. Therefore, we attempted to develop species-specific polymerase chain reactions (PCR) for four pathogenic <em>Aeromonas</em> species. The results of these PCRs were completely consistent with those of <em>rpoB</em> gene analysis. These PCR-based methods can permit the identification of pathogenic <em>Aeromonas</em> species, especially <em>A. dhakensis</em>, more quickly and simply than conventional methods.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107142"},"PeriodicalIF":1.7,"publicationDate":"2025-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143927460","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Black ink staining protocol: A cost-effective substitute in quantifying arbuscular mycorrhizal colonization in plant roots","authors":"Yajie Liu,&nbsp;Menghui Yang,&nbsp;Na Li,&nbsp;Yixin Huang,&nbsp;Chunxue Yang","doi":"10.1016/j.mimet.2025.107141","DOIUrl":"10.1016/j.mimet.2025.107141","url":null,"abstract":"<div><div>Arbuscular mycorrhizal (AM) fungi, ubiquitously distributed across diverse terrestrial ecosystems, establish symbiotic associations with the majority of vascular plants, fulfilling essential physiological and ecological functions. Mycorrhizal development represents the initiation of host-fungus interactions and serves as a metric for assessing mutualistic efficacy. However, mycorrhizal detection underscores the urgent need to develop cost-effective, efficient, and environmentally benign dyestuff. Therefore, wild-collected and laboratory-grown roots of <em>Medicago sativa</em> were selected. Six reagents including black ink, red ink, acid fuchsin, trypan blue, Sudan IV, and aniline blue were evaluated in conjunction with computer vision techniques to identify optimal one. Concurrently, root characteristics were quantified, and interrelationships among root traits, image quality, and colonization indices were analyzed to unravel the mechanism of their interactions. The findings demonstrated that wild roots exhibited pronounced lignification, achieving a mycorrhizal colonization rate of 100 %, which was better than the two laboratory groups. And the fungal community displayed a markedly greater colonization intensity compared to the <em>Claroideoglomus etunicatum</em>. Evaluation of the six reagents revealed distinct staining efficacy, with significant variations in image clarity, gray-level co-occurrence matrix (GLCM) indices, and colonization parameters across treatments. Specifically, aniline blue proved ineffective, while Sudan IV showed selective binding. Notably, black ink in glacial acetic acid achieved optimal mycorrhizal detection efficacy. Moreover, correlation matrix identified microscopic image quality as critical determinant of quantification accuracy, influenced by both reagent types and root properties, and AvgDiam exerted the most substantial impact (|<em>R</em>| &gt; 0.75).</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107141"},"PeriodicalIF":1.7,"publicationDate":"2025-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143906116","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Evaluating the impact of temperatures and exposure times on probiotics viability under pre- and post- technological processes","authors":"M.E. Morán ,&nbsp;M.P. Martínez ,&nbsp;P.J. Vairoletti ,&nbsp;V.L. Poloni ,&nbsp;L.R. Cavaglieri","doi":"10.1016/j.mimet.2025.107140","DOIUrl":"10.1016/j.mimet.2025.107140","url":null,"abstract":"<div><div>Microorganisms such as probiotic yeasts and lactic acid bacteria are capable of surviving—and in some cases thriving—under challenging conditions, including varying feed compositions, moisture levels, and high temperatures typically encountered during feed processing, such as steam pelleting. The primary objective of this study was to evaluate the effect of different temperatures and exposure times on the viability of yeast- and lactic acid bacteria-based probiotics in aqueous solution. Following this, the probiotics were freeze-dried and incorporated separately into a feed matrix to assess their survival during both simulated and actual pelleting processes. In addition, a comparative analysis was conducted to evaluate the viability of <em>Saccharomyces boulardii</em> RC009 under two different drying methods: freeze-drying and fluidized bed drying.</div><div>All strains evaluated exhibited thermoresistance across the tested temperature range, with yeasts demonstrating greater resistance than bacterial strains. Notably, <em>Saccharomyces</em> spp. and <em>Pediococcus pentosaceus</em> showed the highest thermal tolerance. This enhanced resilience may be attributed to the presence of heat shock proteins (Hsps) and antioxidant defense systems in yeasts, and the production of heat-stable exopolysaccharides (EPS-DPS) in <em>P. pentosaceus</em>. Building on these findings, the freeze-dried probiotics were successfully integrated into a feed matrix and subjected to granulation processes to evaluate their viability post-processing. To our knowledge, this is the first study to systematically assess the impact of temperature and exposure time on probiotic viability during both pre- and post-technological treatments in the context of feed production.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"235 ","pages":"Article 107140"},"PeriodicalIF":1.7,"publicationDate":"2025-04-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143921782","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Analysis of vegetative cells and spore germination in food using flow cytometry","authors":"Souichirou Kawai,&nbsp;Miyo Nakano","doi":"10.1016/j.mimet.2025.107137","DOIUrl":"10.1016/j.mimet.2025.107137","url":null,"abstract":"<div><div>The detection of spore-forming bacteria in food is crucial because they potentially cause spoilage. However, traditional microbial tests are time-consuming and inefficient. Here, we report the use of flow cytometry with 5-cyano-2,3-ditolyl-2H-tetrazolium chloride (CTC) staining for the rapid detection and monitoring of spore germination. In particular, we investigated sporulation and metabolic activity in <em>Paenibacillus</em>, <em>Sporosarcina</em>, and <em>Clostridium</em> spp. and validated our method using real food samples. Our approach enabled the rapid detection of spore-forming bacteria and spore germination followed by outgrowth in food. Flow cytometry is a valuable tool for distinguishing vegetative cells and spores in food samples. In this study, we used flow cytometry with CTC staining to determine spore germination and vegetative cell populations. Our findings revealed that 0.2 % peracetic acid treatment inhibited spore germination, as indicated by decreased metabolically active spore count and increased vegetative cell count detected via flow cytometry. Additionally, flow cytometry enabled rapid and accurate differentiation of spores from vegetative cells, providing quantitative insights into bacterial viability within minutes. These findings indicate that flow cytometry is a reliable and practical tool for real-time microbial monitoring in food safety, offering advantages over conventional culture-based methods.</div></div>","PeriodicalId":16409,"journal":{"name":"Journal of microbiological methods","volume":"232 ","pages":"Article 107137"},"PeriodicalIF":1.7,"publicationDate":"2025-04-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"143891130","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}